This invention relates to an apparatus and method for analyzing a quiescent sample of anticoagulated whole blood. More particularly, this invention relates to an apparatus and method for analyzing the blood sample in a quiescent state in order to provide a white blood cell differential count, a reticulocyte count analysis, an enumeration of nucleated red blood cells, and the ability to detect abnormal nucleated circulating cells, such as cancer cells, which are rare events.
Recent advances in analytical hematology have increased the quantity and quality of information available from a patient""s blood sample. As a result, the medical community""s interest in using patients"" blood samples as a diagnostic tool has also increased, with the most commonly performed test performed on anticoagulated whole blood being the complete blood count, or CBC, which is a suite of tests which may include, in addition to the enumeration of the cellular components and platelets, red blood cell metrics; reticulocyte counts; and the leukocyte differential count (LDC or xe2x80x9cDiffxe2x80x9d) which is the classification of the types of white blood cells present in the blood sample. The general physical properties of the sample, namely various cell or counts must be analyzed using quantitative methods relating to the entire sample. In conventional instrumentation and methods, this requires accurate sample metering and dilution, followed by specialized measurement apparatus. Additionally, the instrument must measure quantitative aspects of the individual cells, which usually involves providing a high dilution of the sample with a subsequent passage of the diluted material through a flow cell which measures the cells using electrical or optical means. Still further, qualitative measurements are used to classify the percentage of the total white blood cells which are composed of specific sub populations. The number of sub-populations depends upon the sophistication of the instrument involved, which may be as little as two or more than seven classifications.
Historically, the differential aspects of the CBC have been performed using separate methods from those used for enumeration. For example, the LDC portion of a CBC was traditionally performed by smearing a small amount of undiluted, blood on a slide, staining the dried, fixed smear, and examining the smear under a microscope. Reasonable results can be gained from such a smear, but the accuracy and reliability of the data depends largely on the technician""s experience and technique. One problem with such smears is that the cells must be killed and fixed, and this precludes many types of supravital stains and analyses whose results depend upon the living cell, such as some cytochemical analyses. In addition, the use of blood smears is labor intensive and cost prohibitive, and is therefore generally not favored for commercial applications.
Another method of performing an LDC uses electrical impedance or optical flow cytometry. Flow cytometry involves passing a diluted blood sample through a small vessel wherein electrical impedance or optical sensors can evaluate the constituent cells as they pass serially through the vessel. The same apparatus may also be used to simultaneously enumerate and provide cell metric data. To evaluate WBC""S, the blood sample must be diluted, and the sample must be treated to mitigate the overwhelming number of the RBC""s relative to the WBC""S. Although more expedient and consistent than the above described smear methods, flow cytometry also possesses several disadvantages. One disadvantage of flow cytometry is the plumbing and fluid controls that are necessary for controlling the flow rate of the diluted blood sample past the sensors. The plumbing in current flow cytometers can, and often does, leak, thus potentially compromising the accuracy and the safety of the equipment. These analyses are also generally incapable of providing any type of morphometric analysis, since an actual image of each cell is not obtained; only the total signal from any given cell may be analyzed. Another disadvantage of many current flow cytometers relates to the accuracy of the internal fluid flow controls and automated dilution equipment. The accuracy of the flow cytometer depends upon the accuracy of the fluid flow controls and the sample dilution equipment, and their ability to remain accurately calibrated. Flow controls and dilution equipment require periodic recalibration. The need for recalibration illustrates the potential for inaccurate results and the undesirable operating costs that exist with many presently available flow cytometers. An article authored by John L. Haynes, and published in Cytometry Supplement 3: 7-17 in 1988 describes the principles of flow cytometry, both impedance and optical, and the application of such a technology to various fields of endeavor. Blood samples being examined in flow cytometers are diluted anywhere from 10:1 to 50,000:1.
Another approach to cellular analysis is volumetric capillary scanning as outlined in U.S. Pat. Nos. 5,547,849; 5,585,246 and others, wherein a relatively undiluted sample of whole blood is placed into a capillary of known volume and thickness and is examined while the blood is in a quiescent state. This technique deals with the presence of the red blood cells by limiting the scanning wavelengths to those to which the red blood cells are relatively transparent, and it requires that the sample be treated so that the red blood cells do not aggregate during the measurement process. Thus, this technique is limited to the use of longer wavelength fluorescence, and there is no provision for the enumeration of reticulocytes or nucleated red blood cells. Additionally, as with flow cytometry, no morphologic information is available from the scans. There are a number of commercial instruments available for performing a CBC or related tests, but those which provide more than a few of the CBC tests quickly become complex, expensive and prone to malfunction. In addition, there are a number of methods proposed for specific hematological tests, but these do not provide all of the clinically useful information which is expected in a CBC.
All of the above methods are generally limited to a single mode of analysis, in that a combination of histochemical staining and cellular morphology is not possible. Having the capability to perform both of these types of tests expands the number of groups which can be recognized by the method.
It would be desirable to have a method and apparatus for examining a quiescent sample of anticoagulated whole blood, which method and apparatus are capable of providing accurate results for a LDC, reticulocyte enumeration and detection of nucleated red blood cells and abnormal circulating nucleated cells, such as cancer cells, and does not require sample fluid flow through the sampling chamber during sample analysis.
This invention relates to a method and apparatus for use in examining and obtaining information from a quiescent substantially undiluted anticoagulated whole blood sample which is contained in a chamber. The phrase xe2x80x9csubstantially undilutedxe2x80x9d as used in connection with this invention describes a blood sample which is diluted by no more than about 1:1, and preferably much less. Generally the only reagents that will be used in performing the method of this invention are dyes, stains and anticoagulants, and these reagents, even if liquid, are not designed to dilute the specimen. The analysis may be performed within a chamber having a fixed depth, as long as the depth supports the formation of red blood cell aggregations and lacunae, which form in a layer having a thickness from about seven to about forty microns, depending upon the hematocrit of the sample. However, having a fixed depth makes it more difficult to analyze samples having a widely varying white cell count, and the simultaneous enumeration of reticulocytes is impossible in this type of chamber. Preferably, a chamber is used which has a varying through plane thicknesses as described below. The several regions in the chamber will create sufficient capillary forces in all regions of the chamber so as to cause spreading of the blood sample throughout the chamber, which ultimately results in a quiescent blood sample in the chamber. The only motion in the blood sample at the time of analysis will be Brownian motion of the blood sample""s formed constituents, which motion is not disabling of the utility of this invention. The apparatus includes a sample-holding chamber which has opposite sample-containment walls, at least one of which is transparent, which walls preferably converge in at least one portion of the chamber. In the preferred embodiment, the through plane thickness of the chamber thus varies in different regions of the chamber. As used in this disclosure, the phrase xe2x80x9cthrough planexe2x80x9d refers to a line of sight which corresponds to the shortest distance between the convergent walls in any region of the chamber. The degree of convergence of the two walls, i.e., the distance between the two walls, at any particular point in the chamber is either known, or it can be measured after the sample has been placed in the chamber, as will be described hereinafter.
The thinnest region in the chamber will be sized so that a monolayer of individual red blood cells present in the sample will form when the chamber is filled with the blood sample. The thickness of this region of the chamber should be between about two and about seven microns, and is preferably about five microns. Thus measurements of red cells"" differential reticulocyte counts can be derived in this region of the chamber.
From the thin portion of the chamber, the chamber thickness increases so as to form progressively thicker regions in the chamber that are used to differentiate and differentially enumerate various white cell types and nucleated red blood cells in the blood sample. The nucleated red blood cells tend to be about the size of small lymphocytes, but can be larger. The thickness of the chamber in this region thereof is typically in the range of between about seven to about forty microns. The chamber is contained in a sample holder into which the blood sample can be drawn.
The sample to be assayed is admixed with a colorant which can be, for example, a fluorescent dye or dyes, and the resultant admixture spreads out in the chamber so as to form a quiescent sample that has a varying thickness due to the convergence of the walls of the chamber. The colorant(s) can be added to the sample prior to admission of the sample into the chamber, or the colorant can be added to the sample while the sample is within the confines of the chamber, such as by dry coating the colorant on walls of the chamber. Regions of interest in the chamber are selectively illuminated by a light source having a selected wavelength, or wavelengths, which causes the colorant in the sample to fluoresce. Regions in the chamber containing the red cell monolayers and the white cells and red cell rouleaux in the blood sample are thus scanned, preferably by an optical instrument, and the results of the scans may be digitized and analyzed. Differential platelet counts with platelets grouped by size and RNA content, as determined by fluorescence, can also be derived in the region of the chamber wherein the red cell rouleaux and lacunae form. Platelets with increased RNA are younger platelets.
This invention provides a method for performing at least a three part differential white blood cell count in a sample of anticoagulated whole blood, which method includes the steps of: providing a sampling chamber that is dimensioned so as to enable the aggregation of the red blood cell population and the separation of individual white blood cells from the red cells in a substantially undiluted anticoagulated whole blood sample which is introduced into the chamber. An admixture of a fluorescing colorant or colorants and the anticoagulated whole blood sample is formed in the sampling chamber. The colorant or colorants are operable to differentially highlight at least three different white blood cell types in the whole blood, and preferable five different white blood cell types. The admixture is allowed to disperse through the chamber so as to form separated quiescent groups of one or more individual white blood cells within open lacunae of plasma in optical working fields in the sample. It should be understood that the white blood cells are by in large excluded from the mass of the red blood cells as the latter aggregate. However, for the purposes of this invention, the white blood cells may lie on top of individual red blood cells of the red cell aggregates and still be detected and evaluated as long as the white cells are visible to the scanning instrument. The fields are optically scanned by performing a field-by-field X-Y-Z scan of the dispersed admixture in the chamber under suitable lighting conditions that will cause at least three and preferably five or more different white cell types to be differentially highlighted by said colorant or colorants. All of the differentially highlighted cells which are detected in the admixture are enumerated, and the enumerated cells are grouped by cell type. The same colorants and light conditions can be used to perform reticulocyte or nucleated red blood cell counts in the blood sample due to the presence of nuclear material in the cells. In the case of the reticulocytes, the nuclear material in the cells constitutes remnants of the cells""nucleus, such as intracellular RNA, and in some cases intracellular DNA. The differentially fluorescing intracellular material present in reticulocytes can be characterized as xe2x80x9cremnants of intracellular nucleated materialxe2x80x9d. The reticulocyte and non-nucleated red cell parameter analyses are performed in the thinnest region of the chamber where individual mature red cells and monolayers of mature red cells, as well as reticulocytes can be expected to be found due to their size.
It is therefore an object of this invention to provide a method and apparatus for use in obtaining differential blood cell counts of certain nucleated blood cells and platelets in a quiescent anticoagulated whole blood sample.
It is an additional object of this invention to provide a method and apparatus of the character described which enables a substantially undiluted whole blood sample to be examined for differential white blood cell subpopulation counts; total white cell subpopulation counts; reticulocyte counts; nucleated red blood cell counts; platelete counts; and detection of abnormal nucleated circulating cells, such as cancer cells.